TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana.

Plant organogenesis requires matching the available metabolic resources to developmental programs. In Arabidopsis, the root system is determined by primary root-derived lateral roots (LRs), and adventitious roots (ARs) formed from non-root organs. Lateral root formation entails the auxin-dependent activation of transcription factors ARF7, ARF19, and LBD16. Adventitious root formation relies on LBD16 activation by auxin and WOX11. The allocation of shoot-derived sugar to the roots influences branching, but how its availability is sensed for LRs formation remains unknown. We combine metabolic profiling with cell-specific interference to show that LRs switch to glycolysis and consume carbohydrates. The target-of-rapamycin (TOR) kinase is activated in the lateral root domain. Interfering with TOR kinase blocks LR initiation while promoting AR formation. The target-of-rapamycin inhibition marginally affects the auxin-induced transcriptional response of the pericycle but attenuates the translation of ARF19, ARF7, and LBD16. TOR inhibition induces WOX11 transcription in these cells, yet no root branching occurs as TOR controls LBD16 translation. TOR is a central gatekeeper for root branching that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin-induced genes.

Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

Jasmonate signaling in the field, part I: elicited changes in jasmonate pools of transgenic Nicotiana attenuata populations.

Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Based on more than 20 years of field research on this eco-genomics model system established in our group, we have developed a genetic and analytical toolbox that allows us to assess the importance of particular genes and metabolites for the survival of this plant in its native habitat. This toolbox has been extensively applied to study the activation of jasmonate signaling after the attack of different herbivore species. Here, we provide detailed guidelines for the analysis, under field conditions, of induced changes in jasmonate pools during insect herbivory. The procedures range from selection and field release of well-characterized transgenic lines for testing the physiological consequences of manipulating jasmonate biogenesis, metabolism, or perception to the metabolic elicitation of chewing herbivore attack and the quantification of the resulting changes in jasmonate fluxes.

Jasmonate signaling in the field, part II: insect-guided characterization of genetic variations in jasmonate-dependent defenses of transgenic and natural Nicotiana attenuata populations.

The introduction of genetically modified plants into natural habitats represents a valuable means to determine organismic level functions of a gene and its effects on a plant's interaction with other organisms. Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Here, we provide detailed guidelines for the analysis, under field conditions, of jasmonate-dependent defense and its impact on the plant's native herbivore community. The procedures are based on the field release of transgenic lines silenced for jasmonate biogenesis, metabolism, or perception to conduct association studies between defense trait expression (secondary metabolite and trypsin proteinase inhibitor accumulation) and insect infestations. Additionally, because some insects have evolved mechanisms to "eavesdrop" on jasmonate signaling when selecting their host plants, we describe how leafhoppers of the species Empoasca, which selectively colonize jasmonate-deficient plants, can be used as "bloodhounds" for identifying natural variations in jasmonate signaling among natural N. attenuata populations.

Diverting the flux of the JA pathway in Nicotiana attenuata compromises the plant's defense metabolism and fitness in nature and glasshouse.

A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression.

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast.

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes.